Conventional gene mutation detection uses a variety of methods such as an OLA (oligonucleotide ligation assay) method in which oligonucleotides are bound to a target and ligated using a DNA ligase (for example, see U.S. Pat. No. 4,988,617 and D. Nickerson, Proc. Natl. Acad. Sci., vol. 87, pp. 8923-8927 (1990)), a TDI (template-directed dye-terminator incorporation) method in which labeled ddNTP is used for single base elongation (for example, see Chen X et al., Nucleic Acids Research, vol. 25, No. 2, pp. 347-353 (1997)), and an invader method (for example, see U.S. Pat. No. 5,985,557). However, the conventional methods have problems of multiplex, cost, versatility, or the like.
There is known a conventional technique which comprises the steps of fixing a large number of DNA probes at a high density on a support such as a slide glass with the surface specially treated, and hybridizing a labeled target or a signal detecting probe to detect signals. However, this technique has the problem that its sensitivity is as low as one tenth of the sensitivity of a conventional Southern blotting method (for example, see Masaaki Muramatsu et al., DNA Microarrays and Current PCR Techniques, Shujunsha Co., Ltd. 85-86, 2000) and that the reaction time is relatively long.
In light of the above problems, the present inventors have proposed a novel isothermal nucleic acid amplification method without using any enzyme (for example, see Japanese Patent No. 3267576). This method utilizes a pair of oligonucleotides each comprising three regions (Honeycomb Probe, referred to as “HCP” hereinafter) in which the three respective regions of a first HCP and a second HCP are designed to be composed of base sequences complementary to each other so that only one region of the first HCP may be hybridized with one region of the second HCP when the both HCPs are reacted. This design makes it possible for a plurality of pairs of the HCPs to hybridize to each other and form an assembly substance by a self-assembly reaction of the HCPs when the pairs of HCPs are reacted (this method for the formation of an assembly substance by the self-assembly reaction is referred to as a PALSAR method hereinafter).